Inhibition of pyrimidine biosynthesis by pyrazofurin and by PALA in murine colon tumors in vivo was demonstrated by measuring pyrimidine ribonucleotide pool sizes by means of HPLC. Each agent alone produced a significant decrease in UTP-CTP pools in colon tumor 26. PALA effectively blocked pyrazofurin-induced intracellular accumulation of orotic acid and orotidine, but the two inhibitors in combination were not more effective than PALA alone. Effects of PALA on pyrimidine nucleotide pools were more pronounced in colon tumor cells than in host tissues (colon, intestine, spleen, and liver). Uridine restored both UTP and CTP pools in PALA-treated murine colon tumors whereas cytidine restored only the CTP pool. PALA in combination with pyrimidine analogs may prove to have selective effects on colon tumors in view of the differential effect of this agent on pyrimidine nucleotide pools in colon tumors and host tissues. Dietary or endogenous uridine may negate inhibitory effects of PALA. Resistance to ara-C was demonstrated in murine colon tumor 36 in vivo. Decreased activity of deoxycytidine kinase was demonstrated in ara-C-unresponsive colon tumor 36. The selection and overgrowth of drug-resistant colon tumor cells during therapy poses a threat to successful therapy of colon cancer as it does in chemotherapy of other types of cancer. The purine analog, 9-beta-D-arabinofuranosyl-2-fluoroadenine (2-F-ara-A), after phosphorylation to 2-F-ara-ATP, inhibits DNA synthesis in colon tumor 36. This agent in combination with other agents is of potential interest in experimental chemotherapy of colon cancer. Nitrosoureas, such as MeCCNU, are used in clinical treatment of colon cancer. This agent is more effective against colon tumor 26 than it is against tumors 36 and 38. Inhibition of DNA synthesis in colon tumors in vivo and in culture were observed, and MeCCNU produced alterations in sucrose-gradient-sedimentation patterns of DNA in cultured colon 26 cells that suggest strand scission or the formation of alkali-labile sites in the DNA. Studies of the biochemical effects of combinations of alkylating agents with analogs and enzyme inhibitors are in progress.